The basal body is a microtubule-organizing center responsible for nucleating the cilium, a cellular structure important for a wide variety of cellular functions such as cell locomotion and sensing the surrounding environment(1, 2). Defects in basal body function have been implicated in a number of human diseases such as Bardet-Biedl syndrome(3). Two components that are conserved in basal bodies in many organisms belong to a family of Ca2+ binding proteins called centrins(9, 13, 14). The centrin family can be divided into two main groups based on sequence similarity to the human centrins, Cen2 and Cen3. The analysis of centrin function at basal bodies shows that they are important for the assembly and maintenance of basal bodies(17-20); however, the studies have largely focused on human Cen2 and those related to it, whereas the function of those in the human Cen3 group remains poorly understood. The ciliate protist Tetrahymena thermophila is being used as a system to study centrin function at basal bodies. Tetrahymena contains two centrins at basal bodies, Cen1, the human Cen2 homolog, and Cen2, the human Cen3 homolog. The majority of studies have focused on Cen1 and the function of Cen2 remains to be determined. In the proposed studies here, the localization and function of Cen2 will be examined using a variety of laboratory methods that have proven successful in studies of Cen1. Initially, the localization of Cen2 will be characterized by immunoelectron microscopy and further characterized by monitoring the loading of Cen2 into basal bodies. The function of Cen2 will be analyzed by examining the cen2 knockout strain for defects in basal body assembly and/or maintenance. Monitoring the loading of other components into basal bodies in the cen2 knockout will provide further characterization of Cen2 function. Since Ca2+ is known to be important for Cen1 function, the role of Ca2+ for Cen2 function will be studied by analyzing Tetrahymena strains that express Cen2 containing mutations that perturb Ca2+ binding. Finally, it is yet to be established if the two major centrin groups have distinct or redundant functions. I propose to study the relationship between these centrins by determining if a centrin knockout strain can be rescued by over expression of the heterologous centrin. The studies described here will add to our understanding of the molecular pathways for basal body assembly and will significantly contribute to our knowledge of this important, basic biological structure.